Two mechanisms have been proposed for the biosynthesis of N-linked oligosaccharide chains in glycoproteins: 1) sequential addition of glycosyl units to a growing chain attached to the protein, as has been shown for 0-glycosidic type chains, and (2) prefabrication of the oligosaccharide chain on a lipid intermediate followed by en bloc transfer to the protein. The enclosed proposal is concerned with determining which of these mechanisms is responsible for the glycosylation of soluble N-linked glycoproteins. It is proposed that the two mechanisms are exprimentally distinguishable, since sequential glycosylation, unlike en bloc transfer, should produce partially glycosylated intermediates. These intermediates are expected to be attached to nascent polypetide chains, since considerable evidence suggests that the nascent chains are the primary sites of glycosylation. The proposal takes advantage of two recently developed techniques, the indirect immunoprecipitation of polysomes and the isolation and analysis of nascent chains to describe the isolation of glycosylated nascent chains for a specific glycoprotein, ovalbumin. Experiments are described in which these purified nascent chains will be used to (1) determine if glycosylation proceeds via sequential or en bloc transfer; (2) analyze the rate of glycosyl transfer; (3) determine the spatial relationship between the site of glycosylation and the site of amino acid polymerization.